Impaired Activated/Memory Regulatory T Cell Clonal Expansion Instigates Diabetes in NOD Mice.

Regulatory T cell (Treg) insufficiency licenses the destruction of insulin-producing pancreatic β cells by auto-reactive effector T cells (Teffs), causing spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice. We investigated the contribution to diabetes of the TCR repertoires of naive regulatory T cells (nTregs), activated/memory Tregs (amTregs), and CD4+ Teffs from prediabetic NOD mice and normal C57BL/6 (B6) mice. NOD mice amTreg and Teff repertoire diversity was unexpectedly higher than that of B6 mice. This was due to the presence of highly expanded clonotypes in B6 amTregs and Teffs that were largely lost in their NOD counterparts. IL-2 administration to NOD mice restored such amTreg clonotype expansions and prevented diabetes development. In contrast, IL-2 administration only led to few or no clonotype expansions in nTregs and Teffs, respectively. Noteworthily, IL-2 expanded amTreg and nTreg clonotypes were markedly enriched in islet-antigen specific TCRs. Altogether, our results highlight the link between a reduced clonotype expansion within the activated Treg repertoire and the development of an autoimmune disease. They also indicate that the repertoire of amTregs is amenable to rejuvenation by IL-2.Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes due to the destruction of insulin-producing β cells by auto-reactive CD4+ and CD8+ T cells (1). Diabetes susceptibility in NOD mice is attributed to genetic defects encoded by the insulin-dependent diabetes (idd) loci including over 40 genes, among which the MHC class II and interleukin-2 (IL-2) genes have major contributions to the pathophysiological immune response leading to diabetes (2). The unique I-Ag7 MHC-II molecule expressed by NOD mice has been reported to bind certain peptides with low affinity, thus impairing the thymocyte negative selection process (3,4). Reduced IL-2 production by activated effector T cells (Teffs) was found in NOD mice and linked to an impairment of Treg numbers and functionality (5-7). An increased diabetes incidence was observed in NOD mice bearing an IL-2 alpha receptor subunit with reduced affinity for IL-2, which was associated with a Treg but not a Teff defect (8). Likewise, the administration of low-dose IL-2 to NOD mice promotes Treg expansion and activation, prevents diabetes onset and even cures diabetic mice (9, 10). Thus, the altered tolerance promoting diabetes in NOD mice could be the result of (i) a defect in thymic selection of Treg and Teff T-cell receptor (TCR) repertoires and/or (ii) an altered Treg fitness due to IL-2 deprivation.Previous studies of the TCR repertoires of NOD mice and type 1 diabetes patients yielded inconsistent results. Some studies showed a restricted TRBV and TRBJ chain usage by CD4+ T cells from pancreatic islets and pancreatic lymph nodes (PLN), indicating that overrepresented islet-specific TCRs preferentially express certain TRBVJ genes (11-13). Conversely, others showed a rather diverse gene usage among islet-infiltrating CD4+ T cells in NOD mice (14), including islet-infiltrating memory CD4+ T cells (15, 16), and no common highly used TRBVBJ combinations in T cells from PLN of type 1 diabetes patients (17). Moreover, NOD Tregs were found to have a restricted TRA repertoire and limited overlap with thymic CD4+ T cells (18, 19), while we found similar TRB diversity between NOD peripheral Tregs and Teffs (20). These somehow conflicting results can be explained by the fact that (i) most of these studies were performed on unsorted CD4+ T cells and thus did not distinguish between regulatory and effector populations and/or (ii) most Treg repertoire analyses were done on a limited fraction of the repertoire by using TCR transgenic NOD mice or by focusing on few rearrangements (18, 19). More importantly, most studies have overlooked the existence of regulatory T cell subsets. Indeed, we previously showed that Tregs could be phenotypically divided into CD4+Foxp3+CD44low CD62Lhigh naive Tregs (nTregs) that have a low turnover, and CD4+Foxp3+CD44highCD62Llow activated/memory Tregs (amTregs) that have a very rapid turnover due to their interaction with their cognate antigens (21). In the healthy B6 mouse background, we showed that amTregs are enriched in deep tissue-draining lymph nodes (LNs) and are characterized by unique clonotype expansions (22). Moreover, we showed little overlap between amTreg and nTreg TRB repertoires (22). Thus, studying amTreg and nTreg TCR repertoires without separating them may be confounding.Here we compared the TCR repertoire of splenic amTregs, nTregs and Teffs of prediabetic NOD and normal B6 mice to investigate whether and how an altered TCR repertoire contributes to diabetes. We found that NOD amTregs have an unexpectedly more diverse repertoire than that of B6 mice, due to the absence of expanded clonotypes. Treatment of prediabetic NOD mice with IL-2, which prevents diabetes occurrence, restored the expansion of clonotypes that are preferentially diabetes-reactive. Thus, the TCR repertoire of amTregs is altered by an IL-2 insufficiency, which instigates diabetes development in NOD mice, and is amenable to therapeutic recovery.